Zong-ng Xia

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant.

The crystal structure of the recombinant trypsin‐solubilized fragment of the microsomal cytochrome b5 from bovine liver has been determined at 1.9 Å resolution and compared with the reported crystal structure of the lipase‐solubilized fragment of the membrane protein cytochrome b5. The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16–Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild‐type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro‐environment of the heme pocket and influencing the stability and the redox potential of the protein. Proteins 2000;40:249–257.

Purification and characterization of trichomaglin ‐ A novel ribosome‐inactivating protein with abortifacient activity

Trichomaglin, a novel ribosome‐inactivating protein, has been isolated from root tuber of a plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The isolation and purification procedure included ammonium sulfate precipitation, Sephadex G‐75 chromatography and CM‐Sephadex C‐50 chromatography. The protein was identified to be homogeneous by SDS‐PAGE and FPLC analysis. Its molecular weight is 24,673 dalton and isoelectric point is 5.8, determined by electrospray ionization mass spectroscopy and isoelectric focusing gel electrophoresis respectively. Trichomaglin can inhibit protein synthesis in rabbit reticulocyte lysate with ID50 of 10.1 nM. When rat ribosome was incubated with trichomaglin, a diagnostic RNA fragment appeared on polyacrylamide gel after ribosomal RNAs were treated with acidic aniline. It was concluded that trichomaglin is an RNA N‐glycosidase. In addition, it has been verified to be an abortifacient protein.

Structure of the F58W mutant of cytochrome b5: the mutation leads to multiple conformations and weakens stacking interactions

Phe58 of cytochrome b5 is involved in stacking interactions with heme and the axial ligand His63. To elucidate the contribution of the stacking interactions to protein stability, the crystal structures of the F58Y and F58W mutants were determined at high resolution. The structure of the F58Y mutant is basically the same as that of the wild-type protein. However, the mutation from Phe58 to Trp58 leads to difficulty in growing single crystals and results in a space-group change; the six molecules in the asymmetric unit form two groups that are related by a non-crystallographic twofold axis. The structure of F58W was determined using molecular replacement by making use of the non-crystallographic symmetry. The F58W mutation gives rise to multiple conformations of six side chains, a peptide linking two of the six residues and the extended propionic acid of the heme. The six molecules in the asymmetric unit of the F58W mutant structure are grouped into two types based on their conformations and one of the six molecules exhibits dual conformations. The stacking interactions are weakened owing to the increase/decrease of the angles between the indole ring of Trp58 and the His63/heme rings, which accounts for the lower stability of F58W compared with the wild-type protein.

Crystallization and preliminary crystallographic studies of trichomaglin, a novel ribosome-inactivating protein

Trichomaglin, a novel ribosome-inactivating protein, has been crystallized in two crystal forms using the hanging-drop vapour-diffusion method. The form A and form B crystals belong to the orthorhombic space group P2(1)2(1)2(1) and the hexagonal space group P6(1) (or P6(5)), respectively. X-ray data have been collected to 3.3 and 2.2 A resolution for the form A and B crystals, respectively.